Seveneasy

The pH value of the undiluted nanoformulation is determined using a pH meter and an electrode system; it was measured by using a Mettler Toledo™ SevenEasy pH meter, the electrode was immersed into the sample and left for 5min without stirring during the measurement at a room temperature to allow the pH value to stabilize. The instrument must be calibrated before the measurement. The electrode was thoroughly washed between samples using a stream of distilled water to remove all traces of the previous sample.

Fresh leaf samples of control and treated plants were washed with deionized water to remove surface-staking electrolytes. Simultaneously, leaves were cut into equal size and immersed into 10 ml of deionized water in closed vials and incubated in gentle shaking at room temperature for 4 h. Electrical conductivity was recorded (L_t) with a conductivity meter (Seven Easy, Mettler Toledo AG8603, Switzerland), and after that, sample vials were incubated at 99°C for 20 min and cooled at room temperature, and final electrical conductivity (L₀) was recorded (Lutts et al., 1996 (link)).
For membrane stability index (MSI), fresh leaf samples were washed thoroughly with deionized water, and then, the experiment was conducted into two sets. In one set, leaves were immersed in 10 ml of deionized water and incubated at 40°C for 30 min. In another set, leavers were immersed in 10 ml of water and incubated at 99°C for 10 min. Electrical conductivity was measured for both the sets of vials (Sairam, 1994 (link)). The percentage of electrolyte leakage and MSI were calculated using the following equation:

To assess the glucose utilization of the three isolates, a D-glucose assay kit (glucose oxidase/peroxidase [GOPOD]; Megazyme, Wicklow, Ireland) was used according to the manufacturer’s instructions [33 (link)]. The three isolates were grown on MRS broth at 28°C for 3 days and sampled at 12-h intervals; the supernatants were collected. Then, 3 mL GOPOD reagent was added to each 100-μL aliquot of supernatant and incubated at 42°C for 20 min. Distilled water was used as the reagent blank solution. After the color reaction, absorbance of the samples was read at 510 nm using a spectrophotometer (Genesys 6; Thermo Electron Corp., Waltham, MA, USA). The pH of the MRS culture medium of the isolates was measured at 4-h or 24-h intervals for 3 days using a pH meter (SevenEasy; Mettler-Toledo, Greifensee, Switzerland).

The viscosities and the pH values of the MEs were measured using by Brookfield viscometer (DV-II + Pro Brookfield, The USA, with a shear rate of 100 rpm and pH meter (Mettler Toledo SevenEasy, Switzerland) at 25 °C, respectively.